This invention relates to an assay cartridge having a plurality of aligned adjacent wells which are useful as the reaction vessels for immuno-chemical reactions involving a solid phase and a liquid phase. The assay cartridge has a filter membrane located between the wells and a waste reservoir. By applying a reduced pressure to the waste reservoir, the liquid phase is drawn through the filter and into the waste reservoir. This enables convenient separation of the solid phase reaction products from liquid phase reaction products. This application is related to U.S. patent application Ser. No. 489,519 filed Apr. 28, 1983, now U.S. Pat. No. 4,652,533 which is incorporated herein by reference thereto.
A number of methods exist for the detection of substances of biological origin. One large class of methodology is the immunoassay, where antigens or haptens and their corresponding antibodies are used to probe the sample for each other. One very important variant of the immunoassay is the solid phase immunoassay. (Cf. Catt et al., J. BIOCHEM, 100: 31c (1966); Catt et al., SCIENCE, 158: 1570 (1967); U.S. Pat. No. 3,646,346 by Catt et al., these references and patents, and subsequently cited references and patents are incorporated herein by reference thereto).
Radioactive atoms, such as .sup.125 I, .sup.131 I, .sup.3 H, and .sup.14 C for example, are commonly utilized as the label in solid phase immunoassays. The resulting solid phase radioimmunoassays are quite sensitive but suffer commonly recognized disadvantages. The radioactive nature of the label subjects the assay to stringent regulatory requirements, results in a relatively short reagent shelf life and poses a waste disposal problem.
In an attempt to overcome the disadvantages of radioimmunoassays, several alternative labeling methods have been developed. Foremost among these are the enzyme immunoassays (EIA, ELISA) where an enzyme replaces the radioactive label. (cf. U.S. Pat. No. 3,551,555 by Schuurs). Enzymes commonly utilized as labels are horseradish peroxidase, alkaline phosphatase, B-galactosidase and glucose oxidase. Enzyme immunoassays have an advantage over radioimmunoassays in that the enzyme labels are very stable and special facilities and instrumentation are not required. However, enzyme immunoassays are generally slower and more tedious to perform than radioimmunoassays.
Luminescent labels have been utilized as an alternative to radioactive or enzyme labels. (cf. U.S. Pat. No. 4,201,763 by Monthony et al.; U.S. Pat. No. 3,992,631 by Harte; U.S. Pat. No. 3,999,948 by Deindoerfer et al.; A. Coons, FLUORESCENT ANTIBODY METHODS; J. Danielli (Editor), GENERAL CYTOCHEMICAL METHODS Vol. 1). Fluorescein is the most commonly used label. Although fluorescence immunoassays possess the ease of use advantage of radioimmunoassays and the reagent stability advantage of enzyme immunoassays, prior art fluorescence immunoassays lack the sensitivity of either radioimmunoassays or enzyme immunoassays. This lack of sensitivity has significance in both research and clinical applications with the result that fluorescence immunoassays have seldom been the assay of choice in these applications.
U.S. Pat. No. 4,652,533, discloses a method of solid phase immunoassay for the quantitation of antigen, hapten or antibody analyte in a liquid sample. The solid phase immunoassay incorporates a luminescent label such as a fluorescent label, a phosphorescent label or an atomic fluorescent label. The solid phase immunoassay utilizes for example (i) a plurality of water insoluble particles of about 10 microns or less in size, or (ii) cells, to which an immunoreactant is attached. The analyte or an analyte containing reaction product is reacted with or in competition with or for the immunoreactant while the particles or cells are in a substantially suspended state. The particles or cells which have, or which through subsequent reaction will have, a luminescent label attached thereto are concentrated by microfiltration to a volume substantially less than the volume of the liquid sample which initially contained the analyte. The luminescence of substantialy all of the luminescent label attached to the concentrated particles or cells is measured.
The assay utilizes a particulate solid phase comprising cells or a plurality of water insoluble particles about 10 microns or less in size (i.e. diameter). Particles may be bacteria, mammalian cell fragments or a polymeric substrate such as, for example, polystyrene latex. Particles may be substantially transparent to a beam exciting the label and to resulting luminescence.
The speed and sensitivity of the assay are enhanced by reacting the analyte (or an analyte containing reaction product) with or in competition with or for the solid phase where the latter is suspended. The large surface area of the particulate solid phase can bring significant quantities of immunoreactants into the reaction. Substantially suspending the solid phase distributes these immunoreactants throughout the liquid medium containing the analyte (or analyte containing reaction product). This enhances rapid and complete reaction involving the analyte or analyte containing reaction product.
The solid phase of the assay may then be concentrated to a volume substantially less than the volume of the liquid sample by microfiltration. This yields a two-fold advantage. First, the analyte may be concentrated prior to quantitation, thereby increasing the sensitivity of the assays by a factor substantially identical to the concentration factor. Second, the volume of the solid phase may be concentrated to a volume where a luminescense detector such as, for example, a front face fluorometer may observe substantially all of the luminescent label.
The above discussed assay is useful for the quantitation of antigen, hapten or antibody analyte or analyte occurring on or attached to cells or other particulate material contained in liquid samples of body fluids such as, for example, serum, plasma, urine, saliva or non-body fluids such as, for example, cell culture media, potable water or waste water. Moreover, many biological substances of interest are present in particulate form in nature. Examples are bacterial antigens and mammalian cell surface antigens. The assay for quantitation of analyte occurring on or attached to cells or other particulate material is directly applicable to these systems. Furthermore, assays may be performed on living cells. Soluble proteins, haptens and viruses may be attached by known methods (cf. U.S. Pat. No. 4,201,763 by Monthony et al.) to microscopic latex particles, prepared by known procedures (cf. D. Blackley (Editor), EMULSION POLYMERISATION (Applied Science Publishers Ltd., Essex, England 1975)).
The foregoing assay illustrates an advance in fluorescence immunoassay methodology. This advanced methodology will be of greatest benefit to research and clinical diagnosis when automated apparatus are available for its practice. There is a need for an assay cartridge suitable for practicing the above methodology.